(i) Field of the Invention
The present invention relates to the use of oligopeptide-coated substrates for a novel rapid testing method for the detection of bacteria and bacterial lipopolysaccharides (L.P.S.) in suitable samples, or for removing LPS pyrogens from aqueous solutions, or for removing LPS endotoxins from wounds. The present invention also relates to a novel such substrate.
(ii) Description of the Prior Art
Bacteria and bacterial lipopolysaccharides cause serious problems in food and medicine. As little as about 200 to about 300 Salmonella bacteria, for example, can cause a serious case of food poisoning.
Many bacterial lipopolysaccharides cause fever in animals or humans; hence, they are often termed "pyrogens". As they are produced within the bacterial cell, they are also called "endotoxins". Lipopolysaccharides are major antigens on the cell surface of gram-negative bacteria.
The prior art has provided many tests for bacterial cells and L.P.S. Some test systems use antibodies to complex with bacteria or bacterial L.P.S. There are immunoassay tests for bacterial cells and lipopolysaccharides. These test systems use antibodies. Antibodies, whether polyclonal or monoclonal, are relatively expensive reagents. They can also be unstable at room temperature. Refrigerated storage must be used to prolong their life. This refrigeration requirement is cumbersome.
The quality of most antibody preparations is subject to variations, from one batch to another, in both purity and overall affinity. This is commercially unacceptable. All batches must have minimum acceptable performance. One bad batch in tens of thousands will cause havoc. Such a defective product could result in an expensive recall of many batches of test kits.
One prior art alternative to antibody tests is culture diagnosis. In such test, samples are taken from foods or solutions, and are inoculated in suitable liquid or solid growth media. After passage of time, the culture is checked for colonies or growth of microorganisms. Objects, e.g. foodstuffs (for example, poultry carcasses) may also be checked by rinsing them with sterile water solutions and culturing the rinse solution.
This method will not detect lipopolysaccharides (or endotoxins), but it only detects viable cells. Viable cells may be destroyed in the culturing procedure, and thus not be detected. The worst problem is that culturing is slow. Up to a week or longer may be required before such culture diagnosis may be completed. Furthermore, culture diagnosis and identification require skilled, educated personnel. This makes the process expensive.
The prior art has also suggested many antibody substrate tests to detect bacterial and lipopolysaccharide contamination. Mattingly et al, in Food Technology, (1985) 39: 90-94, described the use of a combination of a microtiter plate and monoclonal antibody preparation for the enzyme immunoassay of bacterial contamination.
In the screening of foods for Salmonella organisms, there is a need for rapid identification of Salmonella antigens, either in liquid cultures or as colonies on plates. The standard dot blot assay method involving nitrocellulose membranes onto which antigens are blotted provides a convenient format for the screening of food samples for pathogens. However, this method requires that after antigen adsorption onto the membrane, the remaining sites must be completely blocked with proteins to prevent non-specific adsorption of antibody-enzyme conjugate, thus adding to the time required to complete the assay. Furthermore, the membrane is brittle, requiring care in handling a large sheet during transfer and washing.
The meat processing industry is obliged to assay for Salmonella contamination in raw materials and finished products before use or sale. Rapid assays for Salmonella will reduce the deterioration of product quality, and the inventory costs incurred pending such assays. Rapid assays should also permit the early prevention and control of salmonellosis, which is very costly in terms of human misery, lost productivity, medical treatment, recall of products, damage suits and confirmation of the disease. Currently, the rapid detection of viable Salmonella cells in food samples by culture methods involves the inoculation of sample material into a suitable liquid pre-enrichment medium (e.g. nutrient broth or buffered peptone water (BPW)) to allow for the recovery of potentially damaged cells, followed by transfer of the sample to a selective enrichment medium, such as selenite cysteine or tetrathionate broth, to allow for the outgrowth of the salmonellae while suppressing non-Salmonella organisms. The entire procedure requires a minimum of 48 hours to complete. The salmonellae in the pre-enrichment or selective enrichment cultures can then be identified either by: enzyme immunoassay (EIA), biochemical and/or serological tests, or a combination of these methods.
Scott and Barclay, in Vox Sang. (1987) 52: 272-280, devised a method to detect antibodies against hydrophobic lipopolysaccharide from rough mutants of gram-negative bacteria. The method was devised in order to detect antibodies against endotoxins in serum in order to diagnose endotoxemia. The lipopolysaccharide of the mutant gram-negative bacteria lacked serotype polysaccharide o-antigen chains. They, thus, could not bind satisfactorily to polystyrene microtiter plates in the ELISA (enzyme-linked immuno-sorbent assay) detection of cross-reactive immunoglobulin G (IgG) anti-endotoxin antibodies. These rough lipopolysaccharides could not bind to the surface of the plate, due to auto-agglutination. The authors therefore reacted the rough lipopolysaccharide with the antibiotic polymyxin B in solution. The complex precipitated. The precipitated polymyxin B-lipopolysaccharide complex was then plated on the polystyrene plates, and found to be stable. The plated complex could react with serum IgG antibodies to endotoxin.
The above-described method is an antibody test, and does not directly detect bacterial lipopolysaccharide antigen. It is not applicable to the detection of lipopolysaccharide antigens.
Issekutz, in the Journal of Immunological Methods (1983) 61: 275-281, described the use of polymyxin B covalently immobilized onto agarose beads, to remove lipopolysaccharide pyrogens in solution. This was done in a bead-packed column. Such beads are expensive and hard to handle and they must be chemically activated in order to allow the immobilization of the polymyxin. These drawbacks make agarose beads unsuitable for a field test kit.
In man and other animals, many Gram negative bacterial pathogens infecting wounds and open abrasions on the skin release lipopolysaccharide (LPS) endotoxins which may cause inflammation, septic toxaemia and other complications which seriously compromise the infected individual.
Many bacterial lipopolysaccharides cause fever in animals or humans; hence, they are often termed "pyrogens". As they are produced within the bacterial cell, they are also called "endotoxins". Lipopolysaccharides are major antigens on the cell surface of gram-negative bacteria.
Polymyxin B is an antibiotic which is bactericidal to gram-negative bacteria. Polymyxin B is a cyclic peptide with a short peptide side chain acylated at its N-terminus with an eight or nine carbon fatty acid. It is known that polymyxin B, when covalently immobilized onto agarose beads, can bind to lipopolysaccharide pyrogens (see Issekutz, in the Journal of Immunological Methods (1983) 61: 275-281). This author described the use of polymyxin B covalently immobilized onto agarose beads, to remove lipopolysaccharide pyrogens in solution. This was done in a bead-packed column. Such beads are expensive and hard to handle; they must be contained and must be chemically activated in order to allow the immobilization of the polymyxin. Polymyxin is stable between pH 2 and 7, even when boiled. Polymyxin can react with lipopolysaccharide antigens of all gram-negative bacteria, including Salmonella spp, Escherichia coli, Brucella abortus and Campylobacter spp.
As taught in applicant's copending Canadian application Ser. No. 2,017,093 filed May 17, 1990, polymyxin B will, bind directly to any hydrophobic surface, e.g. a macroporous polyester cloth which may be woven or unwoven, or may even be matted fibres, by simple adsorption. Polymyxin B can adsorb hydrophobically to such hydrophobic surface e.g., polyester, via its fatty acid residues. As taught in that pending patent application, polymyxin B will adsorb to woven and non-woven cloth, and matted fibre made of hydrophobic materials. Polymyxin B adsorbs best to polyester, but less satisfactorily with polypropylene cloth, polyaramide cloth, or nylon cloth.
This direct binding of polymyxin B to hydrophobic substrates, e.g. SONTARA 8100 is to be contrasted to the ionic binding taught in copending application Ser. No. 564,950 filed Apr. 22, 1988 now U.S. Pat. No. 5,098,417. In that application, a substrate, at least a part of which is cellulosic, is reacted with selected reagents to provide ionic binding sites thereon, and then in ionic form of a drug, which may be polymyxin B is bound to the cloth ionically. The ionic form of the drug can then be eluted from the ionic binding sites in the cloth by ion exchange with ions in body fluids (in the wound). In this way, the drug exerts its beneficial effect.
Pyrogens are toxic substances which cause fever in humans as well as in other animals, and which display toxicity to many living cell types, e.g. mammalian cells maintained in vitro in cell or tissue cultures. Pyrogens must be removed from solutions or any product destined for injection (e.g. pharmaceuticals, e.g. injectable saline, pharmacological drugs and antibiotics) into humans or animals, or for use in the preparation of culture media for the propagation or mammalian cell or tissue cultures. Gram-negative bacterial lipopolysaccharides (LPS) (also termed "endotoxins"), a major constituent of the outer cell wall, are the main types of pyrogens encountered. Almost all Gram-negative bacteria found in nature produce pyrogenic LPS, including (among numerous others), Escherichia coli and Salmonella species.
Several methods have been developed for the removal of LPS pyrogens from solutions, including one method which is based on the affinity of the antibiotic polymyxin B for the LPS molecule. Issekutz, in the Journal of Immunological Methods (1983) 61: 275-281, described the use of polymyxin B covalently bound to agarose beads to remove LPS pyrogens from solutions in a packed column operation. However, the method for preparing the polymyxin-agarose support is laborious and may be subject to batch variations, since it involves chemical treatment in order to immobilize the polymyxin B on the agarose beads, and the extent and uniformity of the treatment may vary from batch-to-batch. Such method is also costly, since the materials, quality control measures and chemical treatments required are relatively expensive. Indeed, products currently available on the market to remove pyrogens from solutions are costly, and this cost in turn affects the cost of the final products (e.g. pharmaceuticals) from which pyrogens must be removed.
Boehringer Mannheim has now provided polymyxin B-SEPHAROSE, namely polymyxin B bound covalently to SEPHAROSE 4B. This product is said to be available in gel form and is said to be able to remove endotoxin impurities. It is also said that the gel can be regenerated by washing with deoxycholate solutions. This pyrogen removal agent suffers from the same disadvantages as disclosed above with respect to the polymyxin B covalently bound to agarose beads. It is manifest that a commercially-attractive process cannot be conceived using this gel filter.
Alerchek Inc. has also now provided an affinity filter in the form of a derivatized PVC-silica composite with polymyxin B sulfate and an LPS binding co-peptide covalently immobilized to the silica. Since this filter is microporous, filtering is effected using a tangential filter apparatus, which is a more expensive technique.